Bilirubin is a metabolic product of hemoglobin derived from aged erythrocyte and is the main component of bile pigment. Blood bilirubin includes direct bilirubin (conjugate form) and indirect bilirubin (free form). Direct bilirubin, whose propionic acid group on the side chain enzymatically forms an ester bond mainly with glucuronic acid in the liver, is highly water soluble, and reacts directly with a diazo reagent to form an azo coloring matter. Indirect bilirubin, whose propionic acid group is in a free state, is low in the water solubility and reacts with a diazo reagent only in the presence of a reaction accelerator such as alcohol, etc. to form an azo coloring matter.
Measurement values obtained by measuring bilirubin in blood includes a total bilirubin value and a direct bilirubin value. Total bilirubin value is a measurement value of conjugate form and free form in total, obtained by reaction with a diazo reagent in the presence of a reaction accelerator. Direct bilirubin value is a measurement value only of conjugate form, obtained by reaction with a diazo reagent in the absence of a reaction accelerator. Individual bilirubin concentrations of conjugate form and free form can be separately determined from these measurement values to make diagnosis of various liver diseases and diacrisis of jaundice. Thus, the measurement of bilirubin is an important phase of the clinical laboratory tests.
A diazo method for measuring color intensity of azobilirubin formed by the above-mentioned reaction of bilirubin with a diazo reagent is a conventional leading method for measuring bilirubin and has been widely used for diagnosis of various liver diseases.
Another method is to measure bilirubin on the basis of changes in absorbance of bilirubin by allowing an oxidizing agent to act on bilirubin in a sample of living body fluid to oxidize bilirubin. The method for measuring bilirubin, using such an oxidizing agent, includes, for example, a BOD method using bilirubin oxidaze (BOD) as an oxidizing agent, a chemical oxidation method using ferricyanide ions, copper ions, vanadate ions, etc. as an oxidizing agent in place of BOD, etc.
Other methods include, for example, a high performance liquid chromatographical method by high performance liquid chromatography, a film method using a mordant-coated film, etc.
Every one of these methods has both merits and demerits and has been not satisfactory yet.
That is, the diazo method has such problems that the reagent is unstable, that is, effective only about 5 days after the preparation and also ascorbic acid or hemoglobin present in a sample interferes with the measurement values. The BOD method has such problems that use of the enzyme inevitably increases measurement costs and the enzyme is effective only about 2 weeks after the preparation because the enzyme is difficult to be stabilized. The chemical oxidation method has such a disadvantage that use of highly toxic metal ions, etc. inevitably involves a waste water treatment problem and an environmental pollution problem. The high performance liquid chromatographic method or the film method needs a special measurement apparatus in spite of satisfactory measurement values, and has such a disadvantage as poor versatility in simultaneous multi-phasic measurement of a large number of samples like other phases of the clinical laboratory test chemistry.
In the current situation it is desirous to develop a method for measuring bilirubin, using a stable and safe reagent, which is applicable to versatile automatic analyzers and has a good correlation to the conventional method, particularly to the enzymatic method with distinguished characteristics.